Covalent labeling of adenylyl cyclase cytosolic domains with γ-methylimidazole-2′,5′-dideoxy-[γ-<SUP>32</SUP>P]3′-ATP and the mechanism for P-site-mediated inhibition

作     者：Doronin, S Murray, L Dessauer, CW Johnson, RA 

作者机构：SUNY Stony Brook Hlth Sci Ctr Dept Physiol & Biophys Stony Brook NY 11794 USA Univ Texas SW Med Ctr Dept Pharmacol Dallas TX 75235 USA 

出 版 物：《JOURNAL OF BIOLOGICAL CHEMISTRY》 (生物化学杂志)

年 卷 期：1999年第274卷第49期

页      面：34745-34750页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 

基　　金：NIDDK NIH HHS [DK-38828] Funding Source: Medline 

主　　题：腺苷三磷酸/类似物和衍生物 腺苷三磷酸/化学合成 腺苷三磷酸/代谢 腺苷三磷酸/药理学 亲和力标记物/化学合成 亲和力标记物/代谢 叠氮化合物/化学合成 叠氮化合物/代谢 交联试剂/代谢 胞质溶胶/酶学 双脱氧核苷酸类 剂量效应关系 药物 大肠杆菌/代谢 咪唑类/化学合成 咪唑类/代谢 同工酶类 动力学 时间因素 

摘      要：A truncated first cytosolic domain of type V adenylyl cyclase (VC1) and a truncated second cytosolic domain of type II adenylyl cyclase (IIC2) were used alone and in the readily reversible complex (***2) to evaluate interactions with each other and with reversible and irreversible P-site ligands, Enzyme activity was used to assess formation and dissolution of ***2. The data suggest that binding of 2 ,5 -dideoxy-3 -ATP to VC1 and IIC2 prevented formation of ***2 and that 2 ,5 -dideoxy-3 -ATP dissociation occurred slowly. To enable configuration specific cross-linking to the catalytic site, 2 ,5 -dideoxyadenosine 3 -[gamma-(1-methylimidazole)-triphosphate] (gamma-MetIm-2 , 5 -dd-3 -ATP) and 2 ,5 -dd-adenosine 3 -(gamma-azidoanilido)-triphosphate (gamma-azidoanilido-2 ,5 -dd-3 -ATP) were synthesized, the former also as its gamma-P-32-labeled analog. gamma-Azidoanilido-2 ,5 -dd-3 -ATP exhibited an inhibitory potency comparable with that of 2 ,5 -dd-3 -ATP, gamma-MetIm-2 ,5 -dd-[gamma-P-32]3 -ATP labeled the individual VC1 and IIC2 domains comparably and covalently to similar to 20% within 1 h. Formation of ***2 resulted in reduced labeling of VC1 but enhanced labeling of IIC2. The data imply that formation of the catalytically active ***2 complex affects the interaction of each domain with the 2 ,5 -dd-3 -ATP, the binding of which also affects the interaction between the two cytosolic domains, leading to a pseudo irreversible inhibition.